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Remarkable improvement in the regiocomplementarity of a Glycine max epoxide hydrolase by reshaping its substrate-binding pocket for the enantioconvergent preparation of (R)-hexane-1,2-diol
Molecular Catalysis ( IF 3.9 ) Pub Date : 2021-09-01 , DOI: 10.1016/j.mcat.2021.111851
You-Yi Liu 1 , Meng-Di Wu 1 , Xiu-Xiu Zhu 2 , Xia-Ding Zhang 1 , Chen Zhang 2 , Yao-Hui Xu 3 , Min-Chen Wu 1
Affiliation  

E. coli/gmeh3, an E. coli transformant expressing GmEH3, had the best regiocomplementarity (αS = 72.4% and βR = 97.6%) for (S)- and (R)-1,2-epoxyhexanes among five tested aliphatic chain rac-1,2-epoxides (1a5a). To prepare (R)-hexane-1,2-diol (1b) with high eep via enantioconvergent hydrolysis of rac-1a, the regioselectivity coefficient αS of GmEH3 for (S)-1a was improved by reshaping its substrate-binding pocket (SBP). Based on the semi-rational design, Trp102, Ile105, Ile178, Pro187 and Leu189 lining GmEH3’s SBP were identified, each of which was substituted with four residues, respectively. From 17 transformants harboring single-site variants of gmeh3, E. coli/gmeh3W102L, /gmeh3W102I, /gmeh3W102V and /gmeh3P187F were selected, catalyzing the conversion of rac-1a into (R)-1b with obviously enhanced eep values of 59.3–78.3%. Then, three transformants containing double-site variants were constructed by combinatorial mutagenesis of gmeh3P187F separately with gmeh3W102L, gmeh3W102I and gmeh3W102V. Among the three transformants, E. coli/gmeh3W102V/P187F displayed the largest αS of 89.7% with βR of 96.2%. The enantioconvergent hydrolysis of 500 mM rac-1a was conducted using 200 mg/mL wet cells of E. coli/gmeh3W102V/P187F at 25°C for 12 h, affording (R)-1b with 83.1% eep and 91.5% yield. The molecular docking simulation analysis demonstrated that GmEH3W102V/P187F more regiopreferentially attacks Cα in the oxirane ring of (S)-1a than GmEH3.



中文翻译:

Glycine max 环氧水解酶的区域互补性通过重塑其底物结合袋以显着改善(R)-己烷-1,2-二醇的对映收敛制备

E.coli / gmeh3是一种表达Gm EH3 的大肠杆菌转化体,在五个测试中 对 ( S )- 和 ( R )-1,2-环氧己烷具有最佳区域互补性(α S  = 72.4% 和β R = 97.6%)脂肪链外消旋-1,2-环氧化物 ( 1a5a )。为了通过rac - 1a的聚合水解制备具有高ee p的( R )-己烷-1,2-二醇( 1b ) ,Gm EH3的区域选择性系数α S对于( S)- 1a通过重塑其底物结合袋 (SBP) 得到改进。基于半理性设计,色氨酸102,异亮氨酸105,异亮氨酸178,脯氨酸187和Leu 189衬里了Gm EH3的SBP进行了鉴定,其中的每一个与四个残基分别被取代,。从携带gmeh3单位点变体的 17 个转化体中选择大肠杆菌/ gmeh3 W102L、/ gmeh3 W102I、/ gmeh3 W102V和/ gmeh3 P187F,催化rac - 1a转化为(R )- 1b具有明显增强的ee p值 59.3-78.3%。然后,通过gmeh3 P187F分别与gmeh3 W102Lgmeh3 W102Igmeh3 W102V的组合诱变构建了三个含有双位点变体的转化体。在三个转化体中,大肠杆菌/ gmeh3 W102V/P187F显示出最大的 α S为 89.7%,β R为 96.2%。使用 200 mg/mL大肠杆菌湿细胞进行500 mM rac -1a 的对映聚合水解/ gmeh3 W102V/P187F在 25°C 下保持 12 小时,得到 ( R ) -1b,具有 83.1% ee p和 91.5% 产率。分子对接模拟分析证实了Gm EH3 W102V / P187F更regiopreferentially攻击Ç α在(的环氧乙烷环小号) - 1A了Gm EH3。

更新日期:2021-09-01
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